Showing papers in "Journal of Cellular Physiology in 1986"
TL;DR: The ability of heparin or that of hexuronyl hexosaminoglygan sulfate (HHS‐4) to protect basic or acidic fibroblast growth factor (FGF) from acid or heat inactivation has been analyzed and indicates that heparIn and HHS‐4, in addition to protecting FGF from inactivation, also acts at another locus, as yet unidentified.
Abstract: The ability of heparin or that of hexuronyl hexosaminoglycan sulfate (HHS-4) to protect basic or acidic fibroblast growth factor (FGF) from acid or heat inactivation has been analyzed. Both freshly prepared basic and acidic FGF stimulate the growth of baby hamster kidney (BHK-21) cells exposed to medium supplemented with transferrin and insulin. Freshly prepared basic FGF was 10 fold more potent than acidic FGF. The addition of heparin to the medium decreased the potency of basic FGF while it potentiated that of acidic FGF. Upon storage of FGF at -80 degrees C, a decline in potency of both basic and acidic FGF was observed. Heparin, when added to the medium, potentiated their activities, which became similar to that of freshly prepared basic FGF. In order to test whether heparin could protect basic or acidic FGF from inactivation, both mitogens were exposed to acid conditions (1% trifluoroacetic acid, pH 1.08, 2 h) or heat (65 degrees C, 5 min) which inactivate basic or acidic FGF. When exposed to such treatment in the presence of heparin or HHS-4, basic and acidic FGF retained their potency. The effect of heparin and HHS-4 on the bioactivity of basic and acidic FGF is truly of a protective nature, since they had no effect when added after inactivation of the mitogens. Potentiation of the bioactivity of the protected mitogens or of the inactivated one could only be observed when cells were exposed to high heparin or HHS-4 concentrations. This indicates that heparin and HHS-4, in addition to protecting FGF from inactivation, also acts at another locus, as yet unidentified.
849 citations
TL;DR: The basis for the increased albumin permeability following the addition of α‐thrombin appears to be a reversible change in endothelial cell shape with formation of intercellular gaps, as well as no morphological evidence of cell lysis.
Abstract: We studied the effect of thrombin on albumin permeability across the endothelial monolayer in vitro. Bovine pulmonary artery endothelial cells were grown on micropore membranes. Morphologic analysis confirmed the presence of a confluent monolayer with interendothelial junctions. Albumin permeability was measured by the clearance of 125I-albumin across the endothelial monolayer. The control 125I-albumin clearance was 0.273 +/- 0.02 microliter/min. The native enzyme, alpha-thrombin (10(-6) to 10(-10) M), added to the luminal side of the endothelium produced concentration-dependent increases in albumin clearance (maximum clearance of 0.586 +/- 0.08 microliter/min at 10(-6) M). Gamma (gamma) thrombin (10(-6) M and 10(-8) M), which lacks the fibrinogen recognition site, also produced a concentration-dependent increase in albumin clearance similar to that observed with alpha-thrombin. Moreover, the two proteolytically inactive forms of the native enzyme, i-Pr2 P-alpha-thrombin and D-Phe-Pro-Arg-CH2-alpha-thrombin, increased the 125I-albumin clearance (0.610 +/- 0.09 microliter/min and 0.609 +/- 0.02 microliter/min for i-Pr2 P-alpha-thrombin and D-Phe-Pro-Arg-CH2-alpha-thrombin at 10(-6) M, respectively). Since the modified forms of thrombin lack the fibrinogen recognition and active serine protease sites, the results indicate that neither site is required for increased albumin permeability. The increase in albumin clearance with alpha-thrombin was not secondary to endothelial cell lysis because lactate dehydrogenase concentration in the medium following thrombin was not significantly different from baseline values. There was also no morphological evidence of cell lysis. Moreover, the increase in 125I-albumin clearance induced by alpha-thrombin was reversible by washing thrombin from the endothelium. The basis for the increased albumin permeability following the addition of alpha-thrombin appears to be a reversible change in endothelial cell shape with formation of intercellular gaps.
309 citations
TL;DR: The present data indicate that bovine endothelial cells derived from large or small vessels are indistinguishable in so far as their response to growth factors, plasma factors, and substrata are concerned.
Abstract: Bovine adrenal and brain cortex and corpus luteum-derived capillary endothelial cells have been established in culture, taking advantage of their ability to proliferate at clonal density when maintained on extracellular matrix (ECM) coated dishes in the presence of serum supplemented medium. All three cell types formed at confluency a monolayer of small, tightly packed, contact inhibited cells that express factor VIII related antigen. Their proliferative response to basic and acidic FGF when cells were maintained on plastic and exposed to serum supplemented medium was similar to that previously reported for endothelial cells derived from large vessels, with acidic FGF being 30-fold less potent than basic FGF. Their requirement for high density lipoproteins and transferrin in order to proliferate actively when maintained on ECM-coated dishes and exposed to serum-free conditions was also similar to that previously reported for endothelial cells derived from large vessels. Heparin strongly reduced the proliferative response of capillary endothelial cells to either basic or acidic FGF, as well as their response to serum alone, regardless of whether cells were maintained on plastic or on ECM-coated dishes. The present data indicate that bovine endothelial cells derived from large or small vessels are indistinguishable in so far as their response to growth factors, plasma factors, and substrata are concerned.
272 citations
TL;DR: Pro purified from human placenta a basic fibroblast growth factor (FGF)‐like molecule which stimulates the production of plasminogen activator (PA) and collagenase, induces DNA synthesis, produces an increase in motility in cultured bovine capillary endothelial cells, and induces angiogenesis in vivo.
Abstract: We have previously purified from human placenta a basic fibroblast growth factor (FGF)-like molecule which stimulates the production of plasminogen activator (PA) and collagenase, induces DNA synthesis, produces an increase in motility in cultured bovine capillary endothelial (BCE) cells, and induces angiogenesis in vivo. The ability of basic FGF to stimulate PA production in BCE cells was used as an assay for the presence of basic FGF-like molecules in extracts of both normal and tumor-derived cultured cells. The identity of the PA-stimulatory activity with basic FGF was confirmed by its high affinity for heparin and by its cross-reactivity with antibodies to human placental basic FGF. Basic FGF-like molecules were identified in eight of ten cell lines tested, and the amount of FGF-like activity present in these cells bore no relation to their origin from normal or tumor tissue. The test cells, BCE cells, had one of the highest levels of FGF-like activity, suggesting that it may have an autocrine role in these cells.
246 citations
TL;DR: The results suggest that thrombin‐induced neutrophil chemotaxis and aggregation are mediated by different mechanisms, sinceChemotaxis is a catalytically independent response whereas aggregation is an active site independent response.
Abstract: Thrombin-induced neutrophil chemotaxis and aggregation were studied using cells isolated from either human or sheep blood. Sheep neutrophils (10(8) cells/ml) exhibited maximum chemotactic migration towards 10(-8)M human alpha-thrombin, 10(-8)M gamma-thrombin (which lacks the fibrinogen site), and 10(-12)MD-Phe-Pro-Arg-CH2-alpha-thrombin (catalytically inactive thrombin). Chemotactic responses of the same magnitude were obtained with human neutrophils (10(8) cells/ml). The chemotactic responses to thrombin were comparable to those obtained with diluted (1:200 v/v) zymosan activated serum (ZAS) and 10(-11)M FMLP. Premixing of the thrombin forms with hirudin in 1:1 stoichiometric amounts abolished the chemotaxis but not chemokinesis Aggregatory responses of human and sheep neutrophils were comparable for ZAS, alpha-thrombin, and gamma-thrombin. The responses of both human and sheep neutrophils to D-Phe-Pro-Arg-CH2-alpha-thrombin were attenuated, indicating that the proteolytic site may be involved in the aggregatory response. The results suggest that thrombin-induced neutrophil chemotaxis and aggregation are mediated by different mechanisms, since chemotaxis is a catalytically independent response whereas aggregation is an active site independent response.
197 citations
TL;DR: Results indicate that GSH may be important in the regulation of cellular proliferation and treatments that prevent the early increase in intracellular GSH levels do not affect protein synthesis but result in a reversible dose‐dependent decrease in the percent of cells capable of entering S phase.
Abstract: The relationship between total glutathione (GSH) content and cell growth was examined in 3T3 fibroblasts. The intracellular GSH level of actively growing cultures gradually decreases as these cells become quiescent by either serum deprivation or high cell density. Upon mitogenic stimulation of sparse, quiescent (G0/G1) cultures with serum, there is a rapid 2.3-fold elevation in intracellular GSH levels which is maximal by 1 h and returns to baseline by 2 h. This is followed by a more gradual increase in GSH content as cells enter the S phase. In addition, the elevation in GSH content is required for maximum induction of DNA synthesis. Treatments that prevent the early increase in intracellular GSH levels do not affect protein synthesis but result in a reversible dose-dependent decrease in the percent of cells capable of entering S phase. These results indicate that GSH may be important in the regulation of cellular proliferation.
171 citations
TL;DR: It is concluded that activation of purinergic receptors increases intracellular free calcium in endothelial cells, and that this increase is probably an essential trigger for synthesis of prostacyclin and the labile endothelium‐derived relaxant factor.
Abstract: The release of vasodilating substances from the vascular endothelium has been postulated to depend on a rise in the level of intracellular free calcium (Cai++). We measured Cai++ in intact monolayers of calf endothelial cells, grown in culture, that were loaded with the fluorescent calcium indicator quin 2. Fluorescence (excitation wavelength 340 nm, emission wavelength 492 nm) was calibrated by raising Cai++ to a maximum with the calcium ionophore ionomycin (0.1 microM) and by lowering it to a minimum with ionomycin plus manganese (0.4 mM), which quenches quin 2 fluorescence completely. Loss of fluorescent dye from the cells was calculated from fluorescence at the isosbestic excitation wavelength (365 nm). Resting Cai++ was 71 +/- 3 (SEM) nM. ATP (adenosine-5'-triphosphate) raised Cai++ dose-dependently and reversibly to 458 +/- 60 nM at a concentration of 10 microM, and at 0.1 mM to values close to those that occurred under ionomycin. ADP (A-5'-PP) and AMP (A-5'-P) had smaller effects with a maximal Cai++ of 287 +/- 72 nM at 30 microM ADP and 176 +/- 17 nM at 0.1 mM AMP. At these concentrations, ADP and AMP attenuated significantly the increase of Cai++ under ATP (10 microM). Adenosine (0.1 or 0.3 mM) and acetylcholine (0.1 to 30 microM) enhanced Cai++ inconsistently, by a maximum of 50 nM. These effects were abolished by theophylline and atropine, respectively. In the absence of extracellular calcium, ATP still raised Cai++, although endothelial responsiveness declined after repetitive stimulations. We conclude that activation of purinergic receptors increases intracellular free calcium in endothelial cells, and that this increase is probably an essential trigger for synthesis of prostacyclin and the labile endothelium-derived relaxant factor.
167 citations
TL;DR: Findings indicate that the activation of protein kinase C and mobilization of Ca2+ evoked by PMA and A23187 respectively are synergistically effective for eliciting a full physiological response of EC in the generation and release of O2−.
Abstract: In order to study the signal transduction mechanism of human endothelial cells (EC), the regulation of superoxide anion (O2−) release in EC has been investigated using the calcium ionophore A23187 and phorbol myristate acetate (PMA), a potential activator of the Ca2+ activated, phospholipid-dependent protein kinase, designated “protein kinase C.” PMA enhanced O2− release from EC, and this enhancement occurred regardless of the presence or absence of extracellular Ca2+. A similar increase was produced by A23187; omission of extracellular Ca2+ prevented this increase. Simultaneous stimulation with PMA and A23187 produced a large increase in O2− release at submaximal concentrations of these agents, which, when added separately, caused minimal effects. These findings indicate that the activation of protein kinase C and mobilization of Ca2+ evoked by PMA and A23187 respectively are synergistically effective for eliciting a full physiological response of EC in the generation and release of O2−.
163 citations
TL;DR: In vitro, the effects of the electrical field produced by constant current on freshly isolated rabbit osteoclasts and well characterized clonal rat osteoblastlike cells showed that different cell types from the same tissue can respond differently to the same electrical signal.
Abstract: We have investigated in vitro the effects of the electrical field produced by constant current on freshly isolated rabbit osteoclasts and on well characterized clonal rat osteoblastlike cells. At field strengths of 0.1 and 1 V/mm, the osteoclasts migrated rapidly toward the positive electrode, whereas the osteoblastlike cells migrated in the opposite direction, toward the negative electrode. Thus, different cell types from the same tissue can respond differently to the same electrical signal. These results have important implications for hypotheses concerning the cellular mechanism of galvanotaxis, and may also clarify the cellular basis of the clinical application of electrical stimulation of bone healing.
156 citations
TL;DR: Using a co‐culture system, a plateau in which the state of differentiation was stabilized could be generated for co‐cultured hepatocytes isolated from adult rat and a disappearance of the initial expression of α1‐fetoprotein and the increase and/or maintenance of albumin secretion were measured.
Abstract: Sinusoidal cells isolated from adult rat liver have been established in primary culture and in cell line. The presence of factor VIII R:Ag and peroxidatic/phagocytosis activities were the criteria used to distinguish in freshly isolated cells the endothelial cells from the Kupffer cells and suggested the endothelial origin of the cell line. Using a co-culture system, the effect of sinusoidal liver cells on hepatocyte functional activity was characterized. A plateau in which the state of differentiation was stabilized could be generated for co-cultured hepatocytes isolated from adult rat and a disappearance of the initial expression of alpha 1-fetoprotein (AFP) and the increase and/or maintenance of albumin secretion were measured with co-cultured hepatocytes isolated from suckling rat. The presence of dexamethasone was required for such beneficial effect. The hepatocyte-stabilizing activity was also produced by a pulmonary endothelial cell line.
147 citations
TL;DR: The demonstration that purified bacterially synthesized GM‐CSF is biologically active in vitro now permits studies to be undertaken on the in vivo effects of this material.
Abstract: Recombinant murine GM-CSF produced in Escherichia coli was purified to homogeneity and tested in parallel with purified native GM-CSF. Both recombinant and native GM-CSF stimulated granulocyte and/or macrophage colony formation by adult and fetal mouse progenitor cells, and with adult marrow cells the specific activity of the recombinant GM-CSF (25 X 10(8) U/mg) was similar to that of the native form (15 X 10(8) U/mg). At high concentrations (greater than 200 U/ml), both forms of GM-CSF also stimulated eosinophil colony formation by adult marrow cells and, at very high concentrations (greater than 800 U/ml), megakaryocyte and some erythroid and mixed-erythroid colony formation. Recombinant GM-CSF was as effective in stimulating the proliferation of the GM-CSF-dependent cell line FD as the native molecule. Both recombinant and native GM-CSF were able to induce partial differentiation in colonies of WEHI-3B myeloid leukemic cells. Recombinant GM-CSF competed effectively for the binding of 125I-labeled native GM-CSF to hemopoietic cells, and antiserum to recombinant GM-CSF also neutralized the biological activity of native GM-CSF. The bacterially synthesized GM-CSF was a slightly more effective stimulus for megakaryocyte colony formation than the native molecule. The demonstration that purified bacterially synthesized GM-CSF is biologically active in vitro now permits studies to be undertaken on the in vivo effects of this material.
TL;DR: The present findings suggest that ras genes can, in certain cells, play a role in promoting differentiation and suppressing proliferation, in contrast to their established oncogenic neoplasiapromoting activity in other cells.
Abstract: Activated mouse N-ras gene transfected into PC12 rat pheochromocytoma cells suppressed proliferation and promoted neuronal differentiation. Normal mouse N-ras in a LTR-containing vector caused differentiation with a reduced efficiency, but normal N-ras in a vector lacking LTR sequences failed to alter the PC12 phenotype. Cultures of NGF-resistant PC12 variant subline U7 also showed outgrowth of neurites and cessation of cell division following transfection with the mutated ras gene. The present findings suggest that ras genes can, in certain cells, play a role in promoting differentiation and suppressing proliferation, in contrast to their established oncogenic neoplasia-promoting activity in other cells.
TL;DR: Results indicate that both Sm‐C and insulin acting through their own receptors increase Leydig cell steroidogenic responsiveness to hCG by increasing hCG receptor number and improving some step beyond cAMP formation.
Abstract: We have investigated the effects of insulin and somatomedin-C/insulinlike growth factor I(Sm-C) in purified porcine Leydig cells in vitro on gonadotrophins (hCG) receptor number, hCG responsiveness (cAMP and testosterone production), and thymidine incorporation into DNA. Leydig cells cultured in a serum-free medium containing transferrin, vitamin E, and insulin (5 micrograms/ml) maintained fairly constant both hCG receptors and hCG responsiveness. When they were cultured for 3 days in the same medium without insulin, there was a dramatic decline (more than 80%) in both hCG receptor number and hCG responsiveness. However the cAMP but not the testosterone response to forskolin was normal. Both insulin and Sm-C at nanomolar concentrations prevent the decline of both hCG receptors and hCG-induced cAMP production. This effect of both peptides was dose dependent with an ED50 of about 1 ng/ml and 5 ng/ml for SM-C and insulin, respectively. Insulin and Sm-C had no additive effect on these parameters. At nanomolar concentrations, Sm-C and insulin enhanced hCG-induced testosterone production but the effect of Sm-C was significantly higher than that of insulin. However, the effect of insulin at higher concentrations (5 micrograms/ml) was significantly higher than that of Sm-C at 50 ng/ml. In contrast, at nanomolar concentrations only Sm-C stimulated [3H]-thymidine incorporation into DNA and cell multiplication, the stimulatory effect of insulin on these parameters, was seen only at micromolar concentrations. These results indicate that both Sm-C and insulin acting through their own receptors increase Leydig cell steroidogenic responsiveness to hCG by increasing hCG receptor number and improving some step beyond cAMP formation. In contrast, the mitogenic effects of insulin are mediated only through Sm-C receptors.
TL;DR: In this paper, the authors describe the purification of a mitogen, termed MGSA for melanoma growth stimulatory activity, from serum-free conditioned medium from the Hs0294 cultures.
Abstract: The Hs0294 human malignant melanoma cell line produces a monolayer mitogen that stimulates the serum free growth of low-density cultures of Hs0294 cells. This report describes the purification of that mitogen, termed MGSA for melanoma growth stimulatory activity, from serum-free conditioned medium from the Hs0294 cultures. MGSA has been purified from acetic acid extracts of lyophilized conditioned medium by gel filtration, reverse-phase high-pressure liquid chromatography (RP-HPLC), and preparative electrophoresis, resulting in a greater than 400,000-fold purification. MGSA bioactivity resides in acid- and heat-stable polypeptides of high and low molecular weight (24-28 kd and less than 14-16 kd). However, the majority of the activity is reproducibly associated with the approximately 16-kd moiety eluting from RP-HPLC at approximately 35% acetonitrile. Reduction with dithiothreitol or B-mercaptoethanol results in a loss of biological activity but does not convert the 24-28-kd moieties to the less than 14-16-kd forms of MGSA. 125I-MGSA that has been purified by preparative electrophoresis (16 kd) specifically binds to Hs0294 melanoma cells and retains 100% of the growth-stimulatory activity. The 16-kd MGSA stimulates the proliferation of Hs0294 cells at concentrations of 0.3-30 pM. The electrophoretic mobility of MGSA is also unaltered by the preparative electrophoresis procedure, further demonstrating that this procedure does not alter the biochemical integrity of the growth factor. Purified MGSA does not enable anchorage-independent growth of normal rat kidney (NRK) cells and is therefore different from the previously described transforming growth factors. The amino acid composition of MGSA differs from that of other previously described growth factors. These data demonstrate that MGSA represents a separate class of growth factors with biological and biochemical properties different from other growth factors.
TL;DR: The data suggest that heparin is slowly internalized by SMC following binding to specific, non‐PF4 dissociable sites, and may accumulate intracellularly and block a crucial point in the proliferative machinery of SMC.
Abstract: The potential of a given amount of heparin to inhibit smooth muscle cell (SMC) proliferation can be increased more than 13 fold if quiescent cultures are pretreated with this mucopolysaccharide for 48 h. The large increase in antiproliferative activity was attributable to a 74% inhibition of the first cell cycle traverse of SMC after serum addition. If the mucopolysaccharide was added to SMC coincident with serum, the initial cell cycle traverse was only suppressed by 27%. In both heparin pretreated and nonpretreated SMC cultures, 48 to 72 h elapsed before substantial inhibition was observed. The inhibitory effects of heparin were reversible and inversely proportional to the starting cell density of the cultures. The effects of known heparin binding proteins on the inhibitory capability of heparin were examined. Neither platelet-derived growth factor (PDGF), low density lipoprotein (LDL), nor platelet factor 4 (PF4) were able to reduce the antiproliferative effects. Heparin retained full biological activity in medium containing serum depleted of all heparin binding proteins by heparin-Sepharose chromatography. These results indicate that heparin does not inhibit growth by preventing serum mitogens or nutrients from interacting with SMC. Rather, our data suggest that heparin is slowly internalized by SMC following binding to specific, non-PF4 dissociable sites. Heparin may accumulate intracellularly and block a crucial point in the proliferative machinery of SMC.
TL;DR: PGE2 enhances both the proliferation and differentiation of osteoblastic cells in vitro, which are probably mediated by two different second messengers dependent on the concentration of PGE2.
Abstract: The effect of prostaglandin E2 (PGE2) on osteoblastic cell proliferation was investigated using osteoblastic clone MC3T3-E1 cells cultured in serum-free medium. PGE2 at 2 micrograms/ml increased the number of the cells by 2 days after its addition. PGE2 raised the level of DNA synthesis in a dose-related fashion after a constant lag time, the maximal effect being at 2-10 micrograms/ml and the level about fourfold over that of the control at 36 hr after its addition. However, at low doses (below 0.2 microgram/ml), PGE2 rather depressed DNA synthesis. Isobutyl methylxanthine counteracted the stimulation of DNA synthesis by PGE2, and forskolin depressed the synthesis, which was inversely correlated with increasing intracellular cAMP content. These results indicate that an increase in cAMP content inhibits DNA synthesis. In addition, 2',5'-dideoxyadenosine did not negate the stimulatory effect of PGE2 on DNA synthesis, suggesting that PGE2 increases DNA synthesis, probably via a pathway different from the adenylate cyclase/cAMP system. Moreover, at a high dose, PGE2 stimulated both the production and degradation of cAMP; the elevation of cAMP content was rapidly depressed by the stimulated degradation system. Consequently, the stimulatory effect of PGE2 on DNA synthesis would be released from the inhibition by cAMP, resulting in an increase in DNA synthesis. Taken together with data from our previous reports, these results indicate that PGE2 enhances both the proliferation and differentiation of osteoblastic cells in vitro, which are probably mediated by two different second messengers dependent on the concentration of PGE2.
TL;DR: H2O2 exposure induces a dose‐dependent disturbance of intracellular calcium homeostatis and the rise in [Ca+++]i is mediated by exposure to H2 O2 in the early phase of the injury, and is not dependent on the continuing presence of the oxidant.
Abstract: The effects of exposure of cultured P388D1 cells to H2O2 on intracellular free calcium ([Ca++]i) was investigated utilizing the intracellular fluorescent calcium chelator "Quin 2." [Ca++]i rose from approximately 150 nM to greater than 2 microM over a time course that was strongly dependent on the concentration of H2O2 used (5 X 10(-5) to 5 X 10(-3) M). After exposure of P388D1 cells to 5 X 10(-3) M H2O2, Quin 2 was fully saturated between 15 and 30 min exposure. During this time, no apparent change in the rate of equilibration of 45Ca++ from the extracellular medium could be detected, whereas in cells preloaded with 45Ca, net 45Ca was lost from the cells at a greater rate than controls. Measurements of total cellular calcium by atomic absorption spectroscopy confirmed that there was a net loss of calcium from the cells during the first 30 min. At time points greater than 45 min after exposure to H2O2 the influx of extracellular 45Ca and net intracellular Ca++, Na+ and K+ rapidly increased. Half times for H2O2 catabolism by the cells varied from about 8 min at 5.0 X 10(-4) M H2O2 to 14.0 min at 5.0 X 10(-3) M. When the total [Ca++]i-buffering capacity of the Quin 2 pool was varied by increasing the loading of intracellular Quin 2 by 68-fold (1.1 X 10(2) - 7.6 X 10(3) amol per cell), the rate of rise of [Ca++]i was depressed by only 1.6-fold following exposure to 5 mM H2O2. During the rise of intracellular [Ca++]i, cell morphology was observed by both light and scanning electron microscopy and revealed that "surface blebs" appeared during this phase of injury. Both the rise in [Ca++]i and "blebbing" were observable before any loss in cell viability was detected by either loss of Trypan blue exclusion or loss of preloaded 51Cr from the cells. From these results we conclude the following, H2O2 exposure induces a dose-dependent disturbance of intracellular calcium homeostatis; the rise in [Ca++]i is mediated by exposure to H2O2 in the early phase of the injury, and is not dependent on the continuing presence of the oxidant; the rate of rise of [Ca++]i is largely independent of the quantity of calcium mobilized to the Quin 2 pool; during the early phase (less than 30 min) of rise of [Ca++]i, only intracellular calcium is involved in the response; these events occur concomitantly with gross morphological changes to the plasma membrane.(ABSTRACT TRUNCATED AT 400 WORDS)
TL;DR: The data suggest that TGF‐β may exert either a growth‐promoting or growth‐inhibiting action on human fetal connective tissues in the presence of other peptide growth factors, which is dependent on fetal age and development.
Abstract: We investigated the influence of transforming growth factor-beta (TGF-beta) on DNA synthesis in human fetal fibroblasts, as measured by the incorporation of [3H]thymidine and cell replication. In serum-free medium, without additional peptide growth factors, TGF-beta had no action on thymidine incorporation. However, in the presence of 0.1% v/v fetal calf serum, TGF-beta exhibited a bi-functional action on the cells. A dose-dependent stimulation of [3H]thymidine incorporation, and an increase in cell number, occurred with fibroblasts established from fetuses under 50 g body weight, with a maximum stimulation seen at 1.25 ng/ml. For fibroblasts from fetuses of 100 g or greater body weight, TGF-beta caused a dose-related decrease in thymidine uptake with a maximal inhibition at 2.5 ng/ml, and a small decrease in cell number. When DNA synthesis was stimulated by the addition of somatomedin-C/insulin-like growth factor I, epidermal growth factor, or platelet-derived growth factor, their actions were potentiated by the presence of TGF-beta on cells derived from fetuses under 50 g body weight, but inhibited on cells obtained from the larger fetuses weighing more than 100 g. Similar results were found for changes in cell number in response to TGF-beta when stimulated by SM-C/IGF I. The ability of TGF-beta to modulate [3H] thymidine incorporation did not involve a change in the time required for growth-restricted cells to enter the S phase of the replication cycle. These data suggest that TGF-beta may exert either a growth-promoting or growth-inhibiting action on human fetal connective tissues in the presence of other peptide growth factors, which is dependent on fetal age and development.
TL;DR: Failure to see any significant differences in cellular toxicity for a larger number of other compounds which either bear limited structural resemblance to cardiac glycosides or interact with the Na+/K+ ATPase in a different manner provides strong evidence that the observed species‐related differences are highly specific for cardiotonic steroids.
Abstract: The relative toxicity of numerous cardiotonic steroids (viz. ouabain, digitoxin, digoxin, convallatoxin, SC4453, bufalin, gitaloxin, digoxigenin, actodigin, oleandrin, digitoxigenin, gitoxin, strophanthidin, gitoxigenin, lanatosides A, B and C, alpha- and beta-acetyl digoxin, alpha- and beta-methyl digoxin) and related compounds towards a number of independent cell lines established from human, monkey, mouse, Syrian hamster, and Chinese hamster have been determined. All cardiac glycosides and their genins, as well as the cardiotonic alkaloid cassaine, exhibited greater than 100-fold higher toxicity towards cultured human and monkey cells in comparison to the cell lines of mouse, Syrian hamster, and Chinese hamster origins. These differences are species-related as all cell lines (both normal as well as transformed) from any one species, as well as cells from the closely related species (e.g., man and monkey or mouse, Chinese hamster, and Syrian hamster), showed similar sensitivity towards these drugs. The failure to see any significant differences in cellular toxicity for a larger number of other compounds which either bear limited structural resemblance to cardiac glycosides (viz. estradiol 17-beta-acetate, testosterone propionate, 21-acetoxy pregnenolone, beta-estradiol, digitonin, tigogenin, and tomatine) or interact with the Na+/K+ ATPase in a different manner (viz. veratridine, sanguinarine nitrate, penicillic acid, vanadium pentoxide, harmaline-HCI,5,5'-diphenyl hydantoin, quindonium bromide, and methyl quinolizinum bromide) provides strong evidence that the observed species-related differences are highly specific for cardiotonic steroids. Studies on the binding of [3H]ouabain show that, in comparison to human and monkey cell lines, no significant binding of the drug is observed in cells derived from the resistant species (i.e., mouse and Chinese hamster). The Na+/K+ ATPase from cells of the resistant species is inhibited at much higher concentrations of ouabain and digitoxin in comparison to the enzyme from human cells, and a good correlation is observed between these concentrations and those reported for inhibition of the enzyme from isolated heart muscles of the same species. These results provide strong evidence that the species-related differences in sensitivity to digitalis have a cellular basis and that the cultured cells from various mammalian species provide a useful model system for investigating the mechanism of action of cardiac glycosides.
TL;DR: Fibronectin and laminin may participate in the regulation of cell populations in vivo and may be involved in epithelial‐mesenchymal interactions.
Abstract: We have examined the effect of laminin and fibronectin on the attachment and growth on type IV collagen of a line of mouse epithelial cells and a strain of adult human fibroblasts. Laminin stimulated attachment of the epidermal cells and fibronectin stimulated fibroblast attachment. At high concentrations (100 micrograms/ml), the attachment proteins altered the growth of cells in culture. The epidermal cells grew better in media containing fibronectin-free serum supplemented with laminin. Fibroblasts, on the other hand, grew best in media containing serum supplemented with fibronectin. These data suggest that laminin promotes epithelial cell growth whereas fibronectin promotes fibroblast growth. This observation was confirmed when these cells were cocultured in the presence of the attachment proteins or of their respective antibodies. The mouse epidermal cells grew best when laminin was added to cocultures of fibroblasts and epithelial cells. Fibroblasts grew best in the presence of antibody to laminin and poorly in the presence of antibody to fibronectin. Thus, fibronectin and laminin may participate in the regulation of cell populations in vivo and may be involved in epithelial-mesenchymal interactions.
TL;DR: Data suggest that the cell volume response of astrocytes in hypoosmotic medium involves the net movement of osmoles by a mechanism dependent on cellular energy and tightly coupled to the (Na, K)‐ATPase ion pump.
Abstract: Cell volume regulation and energy metabolism were studied in primary cultured cerebral astrocytes during exposure to media of altered osmolarity. Cells suspended in medium containing 1/2 the normal concentration of NaCl (hypoosmotic) swell immediately to a volume 40–50% larger than cells suspended in isoosmotic medium. The cell volume in hypoosmotic medium then decreases over 30 min to a volume approximately 25% larger than cells in isoosmotic medium. In hyperosmotic medium (containing twice the normal concentration of NaCl), astrocytes shrink by 29%. Little volume change occurs following this initial shrinkage. Cells resuspended in isoosmotic medium after a 30 min incubation in hypoosmotic medium shrink immediately to a volume 10% less than the volume of cells incubated continuously in isoosmotic medium. Thus, the regulatory volume decrease (RVD) in hypoosmotic medium involves a net reduction of intracellular osmoles. The RVD is partially blocked by inhibitors of mitochondrial electron transport but is unaffected by an inhibitor of glycolysis or by an uncoupler of oxidative phosphorylation. Inhibition of RVD by these metabolic agents is correlated with decreased cellular ATP levels. Ouabain, added immediately after hypoosmotic induced swelling, completely inhibits RVD, but does not alter cell volume if added after RVD has taken place. Ouabain also inhibits cell respiration 27% more in hypoosmotic medium than in isoosmotic medium indicating that the (Na, K)-ATPase-coupled ion pump is more active in the hypoosmotic medium. These data suggest that the cell volume response of astrocytes in hypoosmotic medium involves the net movement of osmoles by a mechanism dependent on cellular energy and tightly coupled to the (Na, K)-ATPase ion pump. This process may be important in the energy-dependent osmoregulation in the brain, a critical role attributed to the astrocyte in vivo.
TL;DR: Actin isotype‐specific probes demonstrated that both β‐ and γ‐cytoplasmic actins are induced by TGFβ, and dose dependent and insensitive to inhibitors of protein synthesis.
Abstract: Stimulation of quiescent cultures of AKR-2B cells with transforming growth factor type beta (TGF beta) resulted in a transitory increase in actin cytoplasmic poly(A) + RNA. Levels of actin mRNA peaked approximately 4-8 hours subsequent to TGF beta addition and approached basal levels by 24 hours. The accumulation of actin transcripts was dose dependent and insensitive to inhibitors of protein synthesis; 1-3 ng/ml TGF beta induced maximal actin gene expression. Actin isotype-specific probes demonstrated that both beta- and gamma-cytoplasmic actins are induced by TGF beta.
TL;DR: The results suggest that the transforming potential of PDGF in an appropriate responsive cell can be controlled not only through its interaction with its own receptor, but also by the presence of inhibitory factors such as TGF‐β.
Abstract: Several growth factors implicated in the process of cellular transformation were tested for their ability to induce anchorage-independent (AI) growth of primary rat embryo (RE) cells. Our results show that in the presence of 10% calf serum, platelet-derived growth factor (PDGF), 1-30 ng/ml, has the strongest effect of all growth factors tested on AI growth. Type-beta transforming growth factor (TGF-beta), by itself, does not stimulate AI growth, and it inhibits the PDGF-induced colony formation in a dose-dependent manner (ED50 approximately 0.03 ng/ml). Qualitatively similar responses are obtained by using an established line of fibroblasts, NIH 3T3 cells; the principal difference between the response of the primary cells and the established cell line is in colony-forming efficiency in soft agar culture (15% and 90%, respectively, for growth of colonies greater than 1,500 micron2 diameter in the presence of 10 ng/ml PDGF). Since AI growth has been shown to correlate well with tumorigenicity in vivo, our results suggest that the transforming potential of PDGF in an appropriate responsive cell can be controlled not only through its interaction with its own receptor, but also by the presence of inhibitory factors such as TGF-beta.
TL;DR: A novel, serum albumin‐binding glycoprotein of molecular weight (mw) 43,000 (43K protein) was initially purified from the culture medium of bovine aortic endothelial (BAE) cells and its secretion by normal mesenchymal cells and by transformed cells of both ectodermal and endodermal origin suggested a general role in cellular function.
Abstract: A novel, serum albumin-binding glycoprotein of molecular weight (mw) 43,000 (43K protein) was initially purified from the culture medium of bovine aortic endothelial (BAE) cells (Sage, H., Johnson, C., and Bornstein, P., J. Biol. Chem. 259:3993–4007, 1984). Its secretion by normal mesenchymal cells and by transformed cells of both ectodermal and endodermal origin suggested a general role in cellular function. To examine the effect of sublethal injury in vitro on the biosynthesis of 43K protein, BAE cells were exposed to endotoxin. At concentrations which produced minimal cell detachment and lysis, the cells secreted 70–100% more protein compared to control cultures, and the relative increase in 43K protein over total protein was approximately three-fold. A second type of cellular injury, manifested by rapid cellular proliferation and migration in response to sparse plating density (a condition that we have termed ‘culture shock’), was also accompanied by a significant increase in the secretion of 43K protein.
Pulse-chase studies revealed that the initial product secreted within 1.5 h was of Mr 38,000, and that between 6 and 21 h this molecule was converted to the final form of Mr 43,000. The 43K protein was not associated with RNA or glycosaminoglycan, but appeared to be linked to complex oligosaccharides containing peripheral sialosyl residues. Treatment with tunicamycin produced lower mw forms that displayed reduced affinity for albumin. By immunologic criteria, peptide mapping, and amino acid analysis, the 43K protein was shown to be structurally distinct from several proteins of Mr 40,000–50,000 associated with endothelium or with serum, including tissue factor, a plasminogen anti-activator, and several apolipoproteins. In addition, the 43K protein was not present in the extracellular matrices of endothelial, fibroblastic, or smooth muscle cells, nor was it found in plasma, serum, platelet releasate, or alveolar lavage fluids.
These studies identify a unique Mr 43,000 glycoprotein that is associated with cellular stress or injury in vitro. As a secreted but nonmatrix macromolecule, this protein may be part of a ‘survival kit’ used by the endothelium to cope with cellular injury.
TL;DR: Studies of a multidrug‐resistant variant of Chinese hamster lung cells selected for resistance to vinca alkaloids revealed marked alterations in transport and intracellular binding of [3H]vincristine compared to parental DC‐3F cells, leading to the conclusion that these two agents most likely do not share the same route for entry in these cells but mightshare the same efflux route.
Abstract: Studies of a multidrug-resistant variant (DC-3F/VCRd-5L) of Chinese hamster lung cells selected for resistance to vinca alkaloids revealed marked alterations in transport and intracellular binding of [3H]vincristine compared to parental DC-3F cells. Influx of [3H]vincristine in DC-3F cells appears to be an equilibrating, but mediated, process. Although saturation kinetics for [3H]vincristine influx were not demonstrated, an extremely high temperature-dependence (Q10 27-37 degrees C = 5-6) and trans-inhibition of influx following preloading of cells with nonradioactive vincristine argue in favor of a carrier-mediated process. Efflux of [3H]vincristine from parental cells conformed to first-order kinetics (t1/2 37 degrees = 3.6 +/- 0.4) and exhibited a lower temperature-dependence (Q10 27-37 degrees C = 3-3.5) than influx. In variant vs. parental cells, influx of [3H]vincristine was reduced 24-fold and efflux was increased two-fold, accounting for the large (approximately 48-fold) reduction in steady-state level of exchangeable drug accumulating in variant cells. Otherwise, transport of [3H]vincristine in these cells showed characteristics similar to parental DC-3F cells. Also, the rate and amount of intracellular binding of [3H]vincristine in variant cells was almost 40-fold lower than in parental cells. These alterations in influx and efflux of [3H]vincristine and its intracellular binding appear to account, at least to a major extent, for the high level of resistance (2,750-fold) of this variant to vinca alkaloids. In contrast, cross-resistance of this variant to daunomycin (178-fold) could be explained only minimally by a transport alteration. Only a two-fold increase in efflux of [3H]daunomycin was demonstrated in variant vs. parental cells along with some decrease in intracellular binding. Influx of [3H]daunomycin was unaltered. In view of these results, we conclude that these two agents most likely do not share the same route for entry in these cells but might share the same efflux route.
TL;DR: It was demonstrated that cells with blebs larger than 50% of the cell diameter did not survive to form colonies and all subcellular organelles, save the ribosomes, were absent from the membrane blebs.
Abstract: Heating synchronous G1 cells at 45.5 degrees C for 3-20 min induced varying degrees of membrane blebbing ranging from nonblebbed cells indistinguishable from control cells to those with blebs larger than the cell itself. Both the proportion of cells exhibiting blebbing and the mean diameter of the blebs increased with heating duration. Scoring individual cells for both blebbing and colony formation demonstrated that cells with blebs larger than 50% of the cell diameter did not survive to form colonies. Electron microscopy showed that all subcellular organelles, save the ribosomes, were absent from the membrane blebs. Freeze fracture replicas revealed no changes in membrane ultrastructure, except on some 15% of the blebs that contained bald patches devoid of membrane particles.
TL;DR: Results indicate that after T GFβ binding the TGFβ:receptor complex becomes rapidly internalized and that TGF β is directed towards lysosomes where it is degraded and released.
Abstract: The fate of 125I-labeled transforming growth factor-beta (125I-TGF beta) after binding to its cells surface receptor has been investigated in BALB/c 3T3 mouse fibroblasts. Binding of 125I-TGF beta to cellular receptors at 4 degrees C is pH-sensitive, being markedly decreased at pH less than 6. Most (approximately 90%) of the 125I-TGF beta bound to cells at 4 degrees C can be removed by a brief treatment with acidic medium but is converted into an acid-resistant state rapidly after shifting the cells to 37 degrees C. Cell-bound 125I-TGF beta is degraded at 37 degrees C and the degradation products are released into the medium. The lysosomotropic bases chloroquine, methylamine, and ammonium and the carboxylic ionophore monensin inhibit the degradation and release of 125I-TGF beta from the cells. Cells allowed to accumulate 125I-TGF beta intracellularly by the action of chloroquine or monensin were treated with the bifunctional agent disuccinimidyl suberate in the presence of detergent Triton X-100; this treatment caused the cross-linking of internalized 125I-TGF beta with the 280-kilodalton TGF beta receptor component. Under conditions in which sustained binding and degradation of saturating 125I-TGF beta concentrations occurs, there is no marked decrease in the binding capacity of the cells even when protein synthesis is blocked with cycloheximide. These results indicate that after TGF beta binding the TGF beta:receptor complex becomes rapidly internalized and that TGF beta is directed towards lysosomes where it is degraded and released. However, the cell surface is replenished with TGF beta receptors recycled after internalization or supplied by a large intracellular pool.
TL;DR: The involvement of the L FA‐1 glycoprotein in T lymphocyte adhesion to endothelial cells suggest that the LFA‐1 molecule may be important in the regulation of leukocyte interactions.
Abstract: Human T lymphocyte adhesion to human endothelial cells is the initial event in T cell migration to areas of extravascular inflammation. The molecular basis for T cell-endothelial cell adhesion was investigated using two different cell-cell adhesion assays: a) a fluorescein cell-cell adhesion assay using nonadherent endothelial cells and fluorescein-labeled T lymphocytes, and b) a radionuclide cell-cell adhesion assay using adherent endothelial cells and 51Cr-labelled T cells. Both assay systems demonstrated comparable quantitative assessment of cell-cell adhesions. The assays were performed at 22 degrees C and adhesions were maximal at 30 min. The results of these adhesion assays confirmed previous reports that T cells adhere to endothelial cells. In addition, we have shown that T cells adhere only marginally to foreskin fibroblasts or bone marrow derived fibroblasts. T cell-endothelial cell adhesions were significantly stronger than either monocytes or B lymphoblastoid cells adhesion to endothelial cells. To demonstrate the molecular mechanisms involved in regulating T cell-endothelial cell adhesions, a panel of function-associated monoclonal antibodies (MAb) were tested for their ability to inhibit T cell adhesion. MAb reactive with the leukocyte surface glycoprotein LFA-1 significantly inhibited T cell-endothelial cell adhesions in both assay systems. In contrast, MAb directed at other surface antigens did not inhibit T cell adhesion. The involvement of the LFA-1 glycoprotein in T lymphocyte adhesion to endothelial cells suggest that the LFA-1 molecule may be important in the regulation of leukocyte interactions.
TL;DR: In this article, 125I-Multi-CSF was radioiodinated to high specific radioactivity (1-4 X 10(5) cpm/ng) with no detectable loss of biological activity and its binding to murine bone marrow cells and factor-dependent cell lines studied.
Abstract: Multipotential colony-stimulating factor (Multi-CSF or interleukin-3) was radioiodinated to high specific radioactivity (1-4 X 10(5) cpm/ng) with no detectable loss of biological activity and its binding to murine bone marrow cells and factor-dependent cell lines studied. Both the native glycosylated molecule purified from a cloned T-cell line (LB-3) and the purified non-glycosylated recombinant molecule produced by E. coli could be radioiodinated. Comparative binding studies with these derivatives demonstrated equal binding affinities and equal numbers of binding sites on various cell types indicating that carbohydrate moieties are not involved in the binding interactions. Binding of 125I-Multi-CSF to several factor-dependent continuous hemopoietic cell lines showed the presence of specific receptors on all cell lines, the receptor number per cell varying from 700 to 13,000 and the apparent dissociation constant from 400 pM to 1 nM. Specific binding of 125I-Multi-CSF was also observed to normal murine hemopoietic cells and the binding to murine bone marrow cells was studied in detail. Bone marrow cells showed 117-130 receptors per cell on average and an apparent dissociation constant of 126-233 pM. However, quantitative autoradiographic analysis indicated that receptors for 125I-Multi-CSF were not distributed randomly on bone marrow cells--nucleated erythroid and lymphoid cells were not labeled while essentially all neutrophilic granulocyte, eosinophilic granulocyte and monocytic cells were labeled. Moreover, in each of the labeled cell lineages grain counts (reflecting receptor number) decreased with increasing maturation and a small subpopulation of marrow cells (0.4-1.5% and including blast cells, monocytes, promyelocytes, and myelocytes) exhibited very high grain counts. The existence of such a subset of marrow cells raises the possibility of functional heterogeneity among marrow cells in their response to Multi-CSF.
TL;DR: Purified human blood neutrophils were able to bind radioiodinated murine granulocyte‐colony‐stimulating factor (G‐CSF) in a specific manner and down‐modulation might be a mechanism whereby these agents activate G‐ CSF receptors and thereby exert some of their effects.
Abstract: Purified human blood neutrophils were able to bind radioiodinated murine granulocyte-colony-stimulating factor (G-CSF) in a specific manner. This factor has previously been shown to stimulate functional activities of human and murine neutrophilic granulocytes and to be functionally analogous to human-derived CSF beta. The binding of 125I G-CSF to human neutrophils was competed for equally by unlabeled G-CSF and CSF beta but not by other CSF's. Saturation analysis indicated that human neutrophils displayed about 700-1,500 receptors for G-CSF/CSF beta per cell. Three other agents (N-formyl-methionine-leucine phenylalanine, bacterial lipopolysaccharide, and human CSF alpha) known to activate neutrophils did not compete directly for G-CSF binding sites but, in preincubation experiments at 37 degrees C, were able to down-modulate the expression of G-CSF receptors on human neutrophils in a dose- and time-dependent manner. This effect was specific since the same agents have been shown elsewhere to up-regulate the expression of other granulocyte surface antigens and other agents were much less effective at down-modulating G-CSF receptors. Since the granulocyte-activating agents increase the sensitivity of human neutrophils to G-CSF/CSF beta and mimic some of the actions of G-CSF on neutrophils, it is suggested that G-CSF receptor down-modulation might be a mechanism whereby these agents activate G-CSF receptors and thereby exert some of their effects.